Small interfering RNA
Encyclopedia
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...
s, 20-25 nucleotides in length, that play a variety of roles in biology. The most notable role of siRNA is its involvement in the RNA interference
RNA interference
RNA interference is a process within living cells that moderates the activity of their genes. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing , and quelling. Only after these apparently unrelated processes were fully understood did it become...
(RNAi) pathway, where it interferes with the expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
of a specific gene. In addition to its role in the RNAi pathway, siRNA also acts in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin
Chromatin
Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
structure of a genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
; the complexity of these pathways is only now being elucidated.
siRNAs were first discovered by David Baulcombe's group at the Sainsbury Laboratory
Sainsbury Laboratory
The Sainsbury Laboratory located at the John Innes Centre in Norwich, Norfolk, England is a world leading institute working on plant-microbe interactions....
in Norwich, England
England
England is a country that is part of the United Kingdom. It shares land borders with Scotland to the north and Wales to the west; the Irish Sea is to the north west, the Celtic Sea to the south west, with the North Sea to the east and the English Channel to the south separating it from continental...
, as part of post-transcriptional
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
gene silencing
Gene silencing
Gene silencing is a general term describing epigenetic processes of gene regulation. The term gene silencing is generally used to describe the "switching off" of a gene by a mechanism other than genetic modification...
(PTGS) in plants. The group published their findings in Science
Science (journal)
Science is the academic journal of the American Association for the Advancement of Science and is one of the world's top scientific journals....
in a 1999 paper titled "A species of small antisense RNA in posttranscriptional gene silencing in plants". Shortly thereafter, in 2001, synthetic siRNAs were shown to be able to induce RNAi in mammalian cells by Thomas Tuschl
Thomas Tuschl
Thomas Tuschl is a German biochemist and Molecular biologist researching RNA.-Biography:Tuschl was born in Altdorf bei Nürnberg. After graduating in chemistry from Regensburg university Tuschl received his PhD in 1995 from Max Planck Institute for Experimental Medicine in Göttingen...
, and colleagues in a paper published in Nature
Nature (journal)
Nature, first published on 4 November 1869, is ranked the world's most cited interdisciplinary scientific journal by the Science Edition of the 2010 Journal Citation Reports...
. This discovery led to a surge in interest in harnessing RNAi for biomedical research
Biomedical research
Biomedical research , in general simply known as medical research, is the basic research, applied research, or translational research conducted to aid and support the body of knowledge in the field of medicine...
and drug development
Drug development
Drug development is a blanket term used to define the process of bringing a new drug to the market once a lead compound has been identified through the process of drug discovery...
.
Structure
siRNAs have a well-defined structure: a short (usually 21-ntNucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
) double-strand RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
(dsRNA) with 2-nt
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
3' overhangs on either end:
Each strand has a 5' phosphate
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...
group and a 3' hydroxyl
Hydroxyl
A hydroxyl is a chemical group containing an oxygen atom covalently bonded with a hydrogen atom. In inorganic chemistry, the hydroxyl group is known as the hydroxide ion, and scientists and reference works generally use these different terms though they refer to the same chemical structure in...
(-OH) group. This structure is the result of processing by dicer
Dicer
Dicer is an endoribonuclease in the RNase III family that cleaves double-stranded RNA and pre-microRNA into short double-stranded RNA fragments called small interfering RNA about 20-25 nucleotides long, usually with a two-base overhang on the 3' end...
, an enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
that converts either long dsRNAs or small hairpin RNA
Small hairpin RNA
A small hairpin RNA or short hairpin RNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA uses a vector introduced into cells and utilizes the U6 or H1 promoter to ensure that the shRNA is always expressed...
s into siRNAs. siRNAs can also be exogenously (artificially) introduced into cells by various transfection
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
methods to bring about the specific knockdown
Gene knockdown
Gene knockdown refers to techniques by which the expression of one or more of an organism's genes is reduced, either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide with a sequence complementary to either an mRNA transcript or a gene...
of a gene of interest. In essence, any gene whose sequence is known can, thus, be targeted based on sequence complementarity with an appropriately tailored siRNA. This has made siRNAs an important tool for gene function and drug target validation studies in the post-genomic era.
RNAi induction using siRNAs or their biosynthetic precursors
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
of an exogenous siRNA can be problematic because the gene knockdown effect is only transient, in particular, in rapidly dividing cells. One way of overcoming this challenge is to modify the siRNA in such a way as to allow it to be expressed
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
by an appropriate vector
Vector (molecular biology)
In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes...
, e.g., a plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
. This is done by the introduction of a loop between the two strands, thus producing a single transcript
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
, which can be processed into a functional siRNA . Such transcription cassettes typically use an RNA polymerase III
RNA polymerase III
RNA polymerase III transcribes DNA to synthesize ribosomal 5S rRNA, tRNA and other small RNAs. The genes transcribed by RNA Pol III fall in the category of "housekeeping" genes whose expression is required in all cell types and most environmental conditions...
promoter (e.g., U6 or H1), which usually directs the transcription of small nuclear RNAs (snRNAs) (U6 is involved in gene splicing
Splicing (genetics)
In molecular biology and genetics, splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation...
; H1 is the RNase
Ribonuclease
Ribonuclease is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the EC 2.7 and 3.1 classes of enzymes.-Function:All organisms studied contain...
component of human RNase P). It is assumed (although not known for certain) that the resulting siRNA transcript is then processed by Dicer
Dicer
Dicer is an endoribonuclease in the RNase III family that cleaves double-stranded RNA and pre-microRNA into short double-stranded RNA fragments called small interfering RNA about 20-25 nucleotides long, usually with a two-base overhang on the 3' end...
.
RNA activation
It has recently been found that dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAaRNAa
Small double-stranded RNA has been found to silence gene expression by an evolutionally conserved mechanism known as RNA interference or RNAi. Such dsRNAs are called small interfering RNAs or siRNA. RNAi can occur at both transcriptional and post-transcriptional levels...
. It has been shown that dsRNAs targeting gene promoters induce potent transcriptional activation of associated genes. RNAa was demonstrated in human cells using synthetic dsRNAs, termed "small activating RNAs" (saRNA
SaRNA
Small activating RNA molecules can induce target gene expression in a phenomenon referred to as dsRNA-induced transcriptional activation ....
s). It is currently not known whether RNAa is conserved in other organisms.
Challenges: avoiding nonspecific effects
Because RNAi intersects with a number of other pathways, it is not surprising that on occasion nonspecific effects are triggered by the experimental introduction of an siRNA. When a mammalian cell encounters a double-stranded RNA such as an siRNA, it may mistake it as a viral by-product and mount an immune response. Furthermore, because structurally related microRNAs modulate gene expression largely via incomplete complementarity base pair interactions with a target mRNAMessenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
, the introduction of an siRNA may cause unintended off-targeting.
Innate immunity
Introduction of too much siRNA can result in nonspecific events due to activation of innate immune responses. Most evidence to date suggests that this is probably due to activation of the dsRNA sensor PKR, although retinoic acid-inducible gene I (RIG-I) may also be involved. The induction of cytokines via toll-like receptor 7 (TLR7) has also been described. One promising method of reducing the nonspecific effects is to convert the siRNA into a microRNA. MicroRNAs occur naturally, and by harnessing this endogenous pathway it should be possible to achieve similar gene knockdown at comparatively low concentrations of resulting siRNAs. This should minimize nonspecific effects.Off-targeting
Off-targeting is another challenge to the use of siRNAs as a gene knockdown tool. Here, genes with incomplete complementarity are inadvertently downregulated by the siRNA (in effect, the siRNA acts as a miRNA), leading to problems in data interpretation and potential toxicity. This, however, can be partly addressed by designing appropriate control experiments, and siRNA design algorithms are currently being developed to produce siRNAs free from off-targeting. Genome-wide expression analysis, e.g., by microarray technology, can then be used to verify this and further refine the algorithms. A 2006 paper from the laboratory of Dr. Khvorova implicates 6- or 7-basepair-long stretches from position 2 onward in the siRNA matching with 3'UTR regions in off-targeted genes.Possible therapeutic applications and challenges
Given the ability to knock down, in essence, any gene of interest, RNAi via siRNAs has generated a great deal of interest in both basic and applied biology. There are an increasing number of large-scale RNAi screens that are designed to identify the important genes in various biological pathways. Because disease processes also depend on the activity of multiple genes, it is expected that in some situations turning off the activity of a gene with an siRNA could produce a therapeutic benefit.However, applying RNAi via siRNAs to living animals, especially humans, poses many challenges. Under experiments, siRNAs show different effectiveness in different cell types in a manner as yet poorly understood: Some cells respond well to siRNAs and show a robust knockdown, whereas others show no such knockdown (even despite efficient transfection
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
).
Phase I results of the first two therapeutic RNAi trials (indicated for age-related macular degeneration, aka AMD) reported at the end of 2005 that siRNAs are well tolerated and have suitable pharmacokinetic properties.
Proof of concept trials have indicated that Ebola-targeted siRNAs may be effective as post-exposure prophylaxis in humans, with 100% of non-human primates surviving a lethal dose of Zaire Ebolavirus, the most lethal strain.
See also
- Oligonucleotide synthesisOligonucleotide synthesisOligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure . The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired...
- EsiRNAEsirnaesiRNA or Endoribonuclease-prepared siRNAs are a mixture of siRNA oligos resulting from cleavage of long double-stranded RNA with an endoribonuclease such as Escherichia coli RNase III or dicer....
- MicroRNA
- ViroidViroidViroids are plant pathogens that consist of a short stretch of highly complementary, circular, single-stranded RNA without the protein coat that is typical for viruses. The smallest discovered is a 220 nucleobase scRNA associated with the rice yellow mottle sobemovirus...
- RNAiAtlas - database of siRNA libraries and their target analysis results
External links
RNAiAtlas provides a siRNA oligonucleotide data from different sources and companies like Dharmacon (ThermoFisher), Qiagen, Ambion, esiRNA for human from Sigma Life Science, and visualize interactions between siRNA oligo and predicted off-target.RNAiAtlas provides a siRNA oligonucleotide data from different sources and companies like Dharmacon (ThermoFisher), Qiagen, Ambion, esiRNA for human from Sigma Life Science, and visualize interactions between siRNA oligo and predicted off-target.
- siRNAdb: a database of siRNA sequences
- DesiRM: Designing of Complementary and Mismatch siRNAs for Silencing a Gene .